This invention relates to recombinant DNA technology for making and using a novel plasmid. More particularly it relates to methods for making and using a de novo synthesized plasmid with defined copy number and selection marker. This de novo synthesized plasmid is useful in making other plasmids and cell strains containing these plasmids. Plasmids are extrachromosomal circular double stranded DNA loops that are transferable from one bacterium to another. Plasmids replicate independently of that of the chromosomal DNA of a host cell. The number of plasmid in a host cell is called the copy number of the plasmid. A given plasmid may be present in a low copy number or a high copy number inside a bacterial cell. The copy number is a genetic characteristic of the replication origin of each plasmid. The replication origin of a plasmid determines its copy number.
The type of replication origin divides plasmids into different incompatibility groups. Different plasmids of same incompatibility group cannot stably coexist in one bacterial cell. On the other hand, plasmids of different incompatibility groups can stably coexist in one bacterial cell. For example, different plasmids of the ColE1-type replication origin (such as plasmids of the family's pBR, pUC and the like) cannot coexist stably in one bacterial cell. Different plasmids of p15A-type replication origin (such as plasmids of the family pACYC and the like) cannot coexist stably in one bacterial cell. However a plasmid of the ColE1-type replication origin and a plasmid of p15A-type origin can coexist stably in one bacterial cell because they belong to different incompatibility groups.
In addition to replication origin, plasmids often contain selection marker genes. A selection marker gene normally allows for phenotypic selection in transformed host cells. Some selection marker genes encode proteins conferring host cell resistance to certain antibiotics. Examples of antibiotics resisted by proteins encoded by selection marker genes include ampicillin, tetracycline, chloramphenicol, kanamycin, gentamycin, rifampicin, spectinomycin, streptomycin, and the like.
Plasmids are useful in DNA cloning, DNA amplification, gene expression, gene therapy, DNA immunization, and like biomedical applications. Substantial efforts have been made to construct numerous plasmids. All of the plasmids made from prior arts are modifications of the plasmids previously obtained from natural sources or recombinant sources. These plasmids inevitably contain some DNA sequences with unknown and/or undesirable function (junk sequences). These junk sequences consume cellular resources and contribute to cellular energy drains. In some cases, the junk sequences have detrimental effects on the plasmid applications. Therefore a novel plasmid, which contains relevant sequences, is generated from de novo synthesis and minimizes the junk sequences, will be valuable in the biomedical applications.